SUPPLEMENTARY INFORMATION de Quimica Fisica and Unidad Asociada CSIC, Universidade de Vigo, 36310,...

In the format provided by the authors and unedited.

Plasmonic nanosensors with inverse sensitivity by means of enzyme-guided crystal growth

Laura Rodríguez-Lorenzo,2 Roberto de la Rica,1,* Ramón Álvarez-Puebla,2 Luis M. Liz-

Marzán,2 Molly M. Stevens1,*

1Department of Materials, Department of Bioengineering and Institute for Biomedical

Engineering, Imperial College London, Exhibition Road, London, SW7 2AZ (UK)

2Departamento de Quimica Fisica and Unidad Asociada CSIC, Universidade de Vigo, 36310,

Vigo (Spain)

Table of contents:

Section S1: Covalent attachment of proteins to gold nanostars

Section S2: Stability of protein-modified gold nanostars

Section S3: Conjugation of GOx to anti-mouse IgG

Section S4: XEDS spectra

Section S5: Vis-NIR spectra for PSA detection via immunoassay.

Section S6: Endogenous levels of PSA in female serum

Section S7: Measuring real samples

References

1 © 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

SUPPLEMENTARY INFORMATIONDOI: 10.1038/NMAT3337

NATURE MATERIALS | www.nature.com/naturematerials 1

http://dx.doi.org/10.1038/nmat3337

Section S1: Covalent attachment of proteins to gold nanostars

Scheme S1. Covalent attachment of proteins to PVP-stabilized Au nanostars showing the

repeating unit of PVP.

The chemical procedure for the modification of PVP-stabilized gold nanostars with proteins

is summarized in Scheme S1. After removing excess layers of PVP (1) by centrifugation and

resuspension in isopropanol (3000 rpm, 3 times), gold nanostars were dispersed in 10 mL of

sodium bicarbonate buffer solution (10 mM, pH 9). Then, 1 mL of 50% glutaraldehyde was

added and the suspension was stirred for 3 h to yield 2.[1] The nanoparticles were then

separated by centrifugation, washed and re-suspended in 10 mL of bicarbonate buffer

solution. Appropriate concentrations of the protein diluted in bicarbonate buffer were reacted

with the nanoparticles dispersion (0.5 mM) containing NaCNBH3 (2 mM) for 3 h at room

temperature to obtain 3.[2] When modifying nanostars with antibodies, non-reacted aldehyde

sites were blocked with bovine serum albumin (BSA, 0.1 mg/mL) and ethanolamine (10

mM) in bicarbonate buffer for 1 h. The protein-modified nanostars were then washed by

centrifugation and re-dispersed in phosphate buffered saline (PBS, 0.01 M phosphate buffer,

0.0027 M potassium chloride, and 0.137 M sodium chloride, pH 7.4, tablets, Sigma).

2

© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.




Figure S1. SERS spectra of: (a) PVP-stabilized gold nanostars, (b) after modification with

glutaraldehyde to yield 2, (c) after covalent attachment of proteins (in this example GOx) to

obtain 3. Inset: amplification of the spectral region between 600 and 200 cm-1.

The formation of 2 and 3 can be confirmed by measuring the surface-enhanced Raman

spectroscopy (SERS) spectra of the nanostars after each modification step, as shown in

Figure S1.[3] The inelastic scattered radiation was collected with a Renishaw Invia Reflex

system, equipped with a two-dimensional Peltier charge-coupled device (CCD) detector and a

confocal Leica microscope. The spectrograph has 1200 g/mm grating with additional band-

pass filter optics. Samples were excited with a 785 nm (diode) laser line. Samples for SERS

were prepared by drop-casting 10 µL of the resulting dispersions on glass slides. Spectra

were collected by focusing the laser line onto the sample by using a 50× objective (N.A.

0.75), providing a spatial resolution of about 1 µm2, with accumulation times of 10 s. The

formation of 2 is proven by the observation of the characteristic peaks for out of plane ring

deformation at 343 and 726 cm-1, H-C=O wagging; ring rocking at 383 cm-1, CH2 ring twisting

at 1196 and 1223 cm-1, CH2 (cyclohexadiene group) wagging at 1343 cm-1, C=C

stretching at 1594 cm-1 and C=C-C=O stretching 1642 cm-1.[4]

The covalent coupling of the protein to the nanostar can be inferred through the spectral

3





changes observed in their corresponding SERS spectra (Figure S1). First after glutaraldehyde

addition the SERS spectrum completely changes its vibrational profile. This is typical of the

generation of high SERS cross-section moieties in the low cross-section aliphatic polymer.

Thus, the spectrum b clearly shows characteristic bands due to the ring including ring

stretching and CCH in plane bendings (region from 1400 to 1600 cm-1) and ring breathings

(996 and 1070 cm-1). Notably, after the protein coupling, the SERS spectrum changes

slightly. The vibrational variations are accumulated in those regions described and are mainly

due to the change in the orientation of the aromatic ring with respect to the plasmonic surface

because of the steric hindrance induced by the protein. Thus, the vibrational change can be

completely explained in full agreement with the surface selection rules.[5] Further, additional

evidence of the coupling is also shown in the change of the relative intensity of the bands

between 1500 and 1600 cm-1. This spectral window contains the contribution of the aldehyde

C=O stretching. Notably, after the coupling of the protein, the relative intensity remarkably

decreases while the vibrational profile varies as a consequence of the disappearance of the

C=O group due to the reductive amination. Further, the new profile shows ring stretching

contributions together with the characteristic amide bands of proteins, especially those of the

amide I at around 1650 cm-1.[6] These spectral features are fully reproducible within the same

sample and in different samples to which further support the covalent coupling of the protein

with no evidence of physisorption.

To confirm that the attachment mainly arises though the formation of covalent bonds and not

only by non-specific adsorption of proteins on the nanostars, the intensity of the bands at 288

and 383 cm-1 was measured in the presence or in the absence of the glutaraldehyde linker.

The band at 288 cm-1 corresponds to the CH2 rocking characteristic of amines; the band at

383 cm-1 was selected for its higher intensity. In Figure S2, the intensity of the bands

increases as the concentration of GOx in the immobilization solution increases when the

process is performed in the presence of glutaraldehyde as described above (red curves).

However, in the absence of glutaraldehyde, only a small increase is registered, which is

4





attributed to minor physisorption of the protein on the nanosensors.

Figure S2. Intensity of the band at 288 cm-1 (a) and 383 cm-1 (b) with respect to the

concentration of GOx when the protein immobilization step is performed in the presence

(red) or in the absence (black) of glutaraldehyde.

5





Section S2: Stability of protein-modified gold nanostars

It is well known that gold nanoparticles can aggregate in solutions containing high salt

concentrations when their surface is not adequately engineered. These aggregation

phenomena can shift the LSPR of the nanosensors to longer wavelengths, therefore

interfering in the detection step. This is particularly worrying when working under

physiological conditions, which usually imply high ionic strength solutions. To prove the

stability of protein-modified gold nanostars in solutions containing high salt concentrations,

the nanoparticles were centrifuged and resuspended in 0.3 M NaCl. In Figure S3, no variation

of the LSPR of the nanosensors is observed, which demonstrates the stability of protein-

modified gold nanostars in solutions containing ions at high concentrations.

Figure S3. Vis-NIR spectra of protein-modified gold nanostars in water (●) and in 0.3 M

NaCl (○).

6





Section S3: Conjugation of GOx to anti-mouse IgG

The conjugation of GOx to anti-mouse IgG was performed by converting amino groups in the

antibody to thiolate groups with 2-iminothiolane followed by conjugation with GOx via the

heterofunctional linker sulfosuccinimidyl 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate

(sulfo-SMCC).[7] To 5 mg of glucose oxidase in 1 mL of PBS buffer pH 7.6, 200 µL of sulfo-

SMCC in water (5 mg/mL) was added two times at 30 min intervals. The reaction mixture

was incubated for 1 hour at room temperature with periodic mixing. Immediately after, the

maleimide-activated GOx was purified with a P10 desalting column using PBS as eluate.

Protein-rich fractions were identified by their characteristic absorbance peak at 280 nm.

Parallel to the enzyme activation, polyclonal anti-mouse IgG developed in goat (Sigma) was

dissolved to a concentration of 1 mg/mL in PBS. Then, 100 µL of 1.5 mg/mL 2-

iminothiolane solution was reacted with the antibody for 1 hour at room temperature. The

resulting thiolated antibody was purified with the desalting column using PBS as eluate; 1

mL fractions were collected and monitored for protein at 280 nm. Finally, the fractions

containing antibody were pooled and immediately mixed with the maleimide-modified

glucose oxidase. After overnight incubation at 4oC, the GOx-modified antibodies were stored

as single-use aliquots at 4 oC until needed.

7





Section S4: XEDS spectra

Figure S4 shows the XEDS spectrum of free-standing silver nanoparticles obtained with 10-14

g·mL-1 GOx. Figures S5, S6 and S7 show XEDS spectra obtained from gold nanostars

modified with 0, 10-20 and 10-14 g·mL-1 GOx, respectively after the signal amplification step.

Each experiment was repeated 10 times at random sites of the grid with identical results.

Figure S4 shows that the free-standing round nanoparticles are made of silver but not gold,

which demonstrates that they are generated via nucleation in solution triggered by the

biocatalytic activity of GOx. In Figure S5, the XEDS spectrum obtained with gold nanostars

that were not modified with GOx after addition of glucose and silver does not show any

signal for silver. This experiment, along with the absence of a sensor response in Figure 2b

(main text), demonstrates that neither glucose nor the proteins that decorate the nanostars are

responsible for the reduction of silver ions, therefore proving that the enzyme-generated

hydrogen peroxide is the reducing agent, as initially hypothesized. Conversely, silver was

detected when the experiment was performed with 10-20 and 10-14 g·mL-1 GOx, which

confirms that the presence of the enzyme, even at ultralow concentrations, is crucial for the

deposition of silver.

8





Figure S4. XEDS analysis of free-standing silver nanoparticles found in the solution

containing 10-14 g·mL-1 GOx after the signal amplification step.

Figure S5. XEDS analysis of gold nanostars that were modified with no GOx after the signal

amplification step.

9





Figure S6. XEDS analysis of gold nanostars modified with 10-20 g·mL-1 GOx after the signal

amplification step.

Figure S7. XEDS analysis of gold nanostars modified with 10-14 g·mL-1 GOx after the signal

amplification step.





Section S5: Vis-NIR spectra for PSA detection via immunoassay.

Figures S8 and S9 show representative spectral changes registered during the detection of

PSA in PBS and serum in the inverse sensitivity regime, respectively.

Figure S8. Vis-NIR spectra of gold nanostars after immunodetection of PSA diluted in PBS

to the final concentration of 0 (black), 10-13 (red), 10-14 (green), 10-15 (blue), 10-16 (orange),

10-17 (violet) and 10-18 (grey).

Figure S9. Vis-NIR spectra of gold nanostars after immunodetection of PSA diluted in whole

serum to the final concentration of 0 (black), 10-14 (red), 10-15 (green), 10-16 (blue), 10-17

(orange) and 10-18 (violet).





Section S6: endogenous levels of PSA in female serum

It has been suggested that serum from female donors may contain PSA.[8] The concentration

of endogenous PSA in the serum used in Figure 4b can be estimated from the experiments in

the absence of PSA performed in PBS and serum, as shown in Figure S8 and S9. It should be

noted that this is an approximation because we are not taking into account the contribution of

non-specific interactions in this calculation. After calculating the difference in the LSPR

absorbance position for both cases and interpolating this value in the calibration curve shown

in Figure 4a, we estimate that the serum from the female donor used here contains

approximately 10-18 g·mL-1 PSA. We used the same serum from the same batch extracted

from the same donor for all our experiments.

Section S7: measuring real samples.

In practice, two possible analytical procedures are envisioned. In the first method, the sample

is tested and the signal interpolated in calibration curves similar to those provided in Figure 4.

In the second method, the sample is serially diluted 1:10 several times. These dilutions should

show an inversely proportional relationship with the concentration, as shown in Figure 4.

Although the second methodology is more elaborated, it has several benefits. First, being

inverse sensitivity such a unique phenomenon, the observation of a negative slope would

increase the confidence in the measurement. Second, it increases the dynamic range of the

approach, that is, it allows quantifying those samples whose concentration is higher than the

upper limit of the dynamic range. Finally, it minimizes background effects due to complex

matrices since these potential interferences are also diluted in the process.





References

[1] Kobayashi, K., Okamoto, I., Morita, N., Kiyotani, T. & Tamura, O. Synthesis of the

proposed structure of phaeosphaeride A. Org. Biomol. Chem. 9, 5825-5832 (2011)

[2] Patterson, M. L. & Weaver, M. J. Surface-Enhanced Raman Spectroscopy as a Probe of

Adsorbate-Surface Bonding: Simple Alkenes and Alkynes Adsorbed at Gold Electrodes. J.

Phys. Chem. 89, 5046-5051 (1985)

[3] Rodriguez-Lorenzo, L., Alvarez-Puebla, R. A., de Abajo, F. J. G. & Liz-Marzan, L. M.

Surface Enhanced Raman Scattering Using Star-Shaped Gold Colloidal Nanoparticles. J.

Phys. Chem. C 114, 7336-7340 (2010)

[4] de la Rica, R., Baldi, A., Fernandez-Sanchez, C. & Matsui, A. Selective Detection of Live

Pathogens via Surface-Confined Electric Field Perturbation on Interdigitated Silicon

Transducers. Anal. Chem. 81, 3830-3835 (2009)

[5] Moskovits, M., Dilella, D. P. & Maynard, K. J. Surface Raman-spectroscopy of a number

of cyclic aromatic-molecules adsorbed on silver - Selection-rules and molecular-reorientation

Langmuir 4, 67-76 (1988).

[6] Tuma, R. Raman spectroscopy of proteins: from peptides to large assemblies Journal of

Raman Spectroscopy 36, 307-319 (2005)

[7] G.T. Hermanson in Bioconjugate Techniques Academic Press, Inc., San Diego, 1996,

pp 57-60.

[8] Chang, Y.-F., Hung, S.-H., Lee, Y.-J., Chen, R.-C., Su, L.-C., Lai, C.-S. & Chou, C. Anal.

Chem. 83, 5324–5328 (2011)





SUPPLEMENTARY INFORMATION de Quimica Fisica and Unidad Asociada CSIC, Universidade de Vigo, 36310,...

Documents

Transcript of SUPPLEMENTARY INFORMATION de Quimica Fisica and Unidad Asociada CSIC, Universidade de Vigo, 36310,...